The present invention relates to the use of daylight fluorescent materials as labels for biological molecules. More specifically this invention involves the use of daylight fluorescent materials in assays for antigens, antibodies, receptors, and fragments of nucleic acid macromolecules with particular base sequences.
Living organisms produce a variety of macromolecules with the unusual property of reversible binding to a highly restricted group of other molecules. The bonds formed are of intermediate strength, involving hydrogen bonds, Van der Waals forces and hydrophobic interactions.
Recognition is a term which has been used to describe this general phenomenon. Recognition is utilized widely by the immune system. Receptors on the surfaces of somatic cells recognize the presence of minute quantities of hormones; receptors on neurons and muscle cells recognize neurotransmitters in tissue fluids. Interaction of complementary strands of nucleic acids and of enzymes and their substrates are further examples of this general phenomenon.
Increasingly, recognition has become the preferred method of detecting specific biological molecules for scientific and clinical applications. The specificity and sensitivity of these reactions is very high.
The most common method for detecting biological recognition is to label one of the reacting species with a radioactive isotope as in radioimmunoassays (RIA). This method is effective and is used widely, especially in research laboratories which have the specialized equipment for use and detection of radioisotopes. The health hazard and expense involved in the use of radioisotopes, however, have limited use of the method outside of the research laboratory.
One alternative to the use of radioisotopes as labels is to covalently bind certain enzymes capable of generating a colored species from a colorless substrate to one of the reacting molecules. Although methods involving enzyme labels are very sensitive, time, temperature, and other conditions must be scrupulously controlled in order to achieve accurate results. In addition, enzymes are prone to denaturation and degradation during storage.
Fluorescent labels provide a third alternative and are not subject to the above described limitations. With sophisticated equipment, very high sensitivity can be achieved (U.S. Pat. No. 4,261,968). But fluorescent labels are subject to bleaching over time and under ordinary conditions of measurement.
Therefore despite the advantages of existing methods of assay involving fluorescent labels, there is a need for simpler assay methods. This is particularly true where the assay is to be conducted in an environment where sophisticated instrumentation is unavailable, such as small clinics, the farm or the home. Accordingly, it is desirable to develop assay methods utilizing fluorescent labels which do not require the specialized light sources or detectors otherwise used in such assays. Ideally it is desirable to achieve quantitative or semi-quantitative results using conventional light sources and the unaided eye, in a manner analogous to the use of pH paper.